In the Phase I grant we proposed and fully implemented one aspect of our overall goal of developing advanced instrumentation and reagents for ion and second messenger imaging and analysis. We successfully produced, in production quantities, the fluorescence energy transfer reagent for cyclic AMP imaging, FICRhR, originated at UCSD, of which Atto Instruments, Inc. owns the exclusive world-wide rights. We also developed the hardware, software, and methods to utilize it for the dynamic measurement of cyclic AMP in living cells and simultaneous cAMP and calcium imaging in conjunction with fura-2. In the phase II SBIR project, Atto Instruments plans to 1.) market FICRhR in its current structure and develop improved reagents for imaging CAMP as well as other second messengers, 2.) develop a non-radioactive fluorescence assay for cAMP in cell extracts using FICRhR, and 3.) develop the hardware and software for ultra high speed microscopic ratio imaging and photometry incorporating a novel excitation light source which can switch between two wavelengths at rates higher than 5 Khz. Basic and clinical research into a myriad of cell functions, both in health and disease, regulated by cyclic AMP, calcium, and other second messengers will be advanced by the commercial availability of these important investigative tools. PROPOSED COMMERCIAL APPLICATION: Support in this project will enable us to: 1) optimize, improve, and commercialize FIRChR ,the only available fluorescent sensor for imaging cyclic AMP in living cells. 2) develop additional products based upon FIRChR for greater sensitivity, dynamic range, fluorescent stability, method of application, and the non- radioactive fluorescence assay of cyclic AMP in cell extracts. 3.) develop hardware/software products for ultra high speed ratio imaging and photometric detection of calcium (fura-2), cyclic AMP (FICRhR), and other fluorescent probes in living cells.